Trypanosomatids constitute a group of parasitic protozoa of major medical importance. A recent report of the World Health Organization estimates that over a million people succumb to diseases brought on by these organisms each year and much greater numbers are at constant risk. In addition, animal trypanosomiasis in sub-Saharan Africa contributes to a severe deficit of milk and other cattle products. Consequently, these organisms represent a major public health challenge and understanding their biology is an important international priority. Chromosomal DNA replication is an unexplored aspect of trypanosome biology. Two themes animate our interest in this vital cellular process and make it one of topical interest. Recent experiments, carried out in organisms as diverse as yeast and Drosophila, clearly sustain the notion that elements and factors controlling DNA replication are intimately involved in the epigenetic control of gene expression by affecting higher order chromatin assembly. This issue is emerging as a rich vein for further research. In a separate development, data arising from the ongoing trypanosomatid genome projects suggest that their chromosomes have an amazing, and hereto unprecedented, large-scale architecture comprising of large, strand-specific transcriptional domains. As few as 2 such domains can cover an entire chromosome (for example, chromosome I in Leishmania mci/or), radiating outwards along opposite strands from a chromosome-internal location, It has been suggested, in this context and elsewhere, that replication origins (oris) may serve to organize chromosomes into such domains We intend to lay the groundwork for an exploration of these issues in trvpanosomatids by defining the sequences controlling DNA replication in Trypanosoma brucei. The experimental approach will involve the use of a panel of autonomously replicating single-copy episomes and defined artificial mini-chromosomes that we have built for this organism. As small, dispensable genetic elements, expected to bear all the cis-acting signals necessary for autonomous replication, these extrachromosomal elements will provide the model system on which we will base our initial studies. As DNA replication is regulated at initiation, a major part of our effort will be devoted to the delineation of art ori on these elements. In addition, preliminary results demonstrate that a region encompassing a transcriptional promoter on these episomes is necessary for plasmid DNA replication. We propose to use linker substitution analysis to precisely map this functional element(s). Delineating the anatomy of key regulatory sequences controlling DNA replication and determining their disposition on chromosomes may provide the groundwork for a deeper analyses of the remarkable global architecture of trypanosome chromosomes or the epigenetic control of gene expression in these organism.